Project Summary Oral squamous cell carcinomas (OSCC) are the most common malignancies of the head and neck region with increasing global incidence, thus constituting a serious public health concern. Despite extensive research, no preventative or therapeutic strategies have been successfully developed to target this potentially lethal form of cancer. This shortcoming is due to a gap in knowledge of the underlying genetic and epigenetic events that contribute to the development and progression of OSCC. Therefore, there is a vital need for mechanistic studies to better dissect the molecular etiology of this disease and to identify new targets for effective preventions and treatments. Evidence from our laboratory and others strongly suggest that the transcription factor p63, specifically the most prevalent DNp63 isoforms, function as an oncogene in OSCC. We have shown that overexpression of DNp63 in the squamous epithelium of transgenic mice results in a severe phenotype that share many of the underlying histological, pathological and molecular features of early stage human SCC. Similarly, others have shown that established OSCCs are exquisitely dependent on p63 for cell survival and growth. However, the molecular underpinnings through which DNp63 functions in the early and late stages of this disease remains relatively ill-understood. Hence, to determine the contribution of DNp63 in the development of OSCC, we will utilize Tet-inducible DNp63 overexpressing transgenic mice (DNp63TG), developed in our laboratory, to address two important areas of interest. In Aim 1, we propose to experimentally evaluate the functional role of DNp63 in the successive stages of oral tumor development by performing 4- Nitroquinolone-1-oxide (4NQO) carcinogenesis studies on the control and DNp63TG mice. In addition, to examine the relative distribution and contribution of the various heterogenous tumor cell populations and to determine global DNp63-driven oncogenic changes, we will transcriptionally profile WT and DNp63TG OSCC tumors by single-cell RNA-sequencing. Data obtained from such in vivo studies will provide a better understanding of the molecular driver of the DNp63 isoform in the early and late stages of OSCC. In Aim 2, we propose to examine the potential causal and synergistic link between DNp63 and one of its downstream targets, the PI3K-Akt signaling pathway, in the development and progression of OSCC in the 4NQO mouse model. In parallel, we will parse the publicly available TCGA, OSCC cell lines, and mouse 4NQO RNA-seq datasets to identify DNp63-specific gene networks and crucial molecular players that might play a conserved role in both human and mouse OSCC. Collectively the information gleaned from these studies will help significantly enhance our understanding of the DNp63-dependent and independent molecular mechanisms that underlie the initiation of preneoplasia and their passage to invasive OSCC. Furthermore, defining the molecular signatures of preneoplasia formation and progression will also unearth new driver pathways and biomarkers for future exploitation as preventative and therapeutic options.